Orally delivered water soluble Coenzyme Q10 (Ubisol-Q10) blocks on-going neurodegeneration in rats exposed to paraquat: potential for therapeutic application in Parkinson's disease.

BACKGROUND: Paraquat, still used as an herbicide in some parts of the world, is now regarded as a dangerous environmental neurotoxin and is linked to the development Parkinson's disease (PD). Paraquat interacts with cellular redox systems and causes mitochondrial dysfunction and the formation of reactive oxygen species, which in turn, plays a crucial role in the pathophysiology of PD. Various antioxidant therapies have been explored with the expectations that they deliver health benefits to the PD patients, however, no such therapies were effective. Here we have tested the neuroprotective efficacy of a novel water-soluble CoQ10 (Ubisol-Q10), in a rat model of paraquat-induced neurodegeneration in order to evaluate its potential application in the management of PD. RESULTS: We have developed a rat model of progressive nigrostriatal degeneration by giving rats five intraperitoneal injections of paraquat (10 mg/kg/injection), once every five days. Neuronal death occurred over a period of 8 weeks with close to 50% reduction in the number of tyrosine hydroxylase-positive cells. Ubisol-Q10, at 6 mg CoQ10/kg body weight/day, was delivered as a supplement in drinking water. The intervention begun after the completion of paraquat injections when the neurodegenerative process had already began and about 20% of TH-positive neurons were lost. Ubisol-Q10 treatment halted the progression of neurodegeneration and remaining neurons were protected. The outcomes were evaluated based on the number of surviving tyrosine hydroxylase-positive neurons in the substantia nigra region and improved motor skills in response to the Ubisol-Q10 intervention. To maintain this neuroprotection, however, continuous Ubisol- Q10 supplementation was required, if withdrawn, the neuronal death pathway resumed, suggesting that the presence of CoQ10 was essential for blocking the pathway. CONCLUSION: The CoQ10, given orally as Ubisol-Q10 in drinking solution, was effective in blocking the progression of neurodegeneration when administered therapeutically (post-toxin injection), at a much lower concentration than other previously tested oil soluble formulations and well within the acceptable daily intake of 12 mg/kg/day. Such unprecedented neuroprotection has never been reported before. These results are very encouraging and suggest that Ubisol-Q10 should be further tested and developed as a therapy for halting the progression of PD.

Involvement of NOS3 in RA-Induced neural differentiation of human NT2/D1 cells.

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5' regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (-734 to -989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.

Effects of Tolerance-Induced Preconditioning on Mitochondrial Biogenesis in Undifferentiated and Differentiated Neuronal Cells.

BACKGROUND: Mitochondrial biogenesis occurs in response to chronic stresses as an adaptation to the increased energy demands and often renders cells more refractive to subsequent injuries which is referred to as preconditioning. This phenomenon is observed in several non-neuronal cell types, but it is not yet fully established in neurons, although it is fundamentally important for neuroprotection and could be exploited for therapeutic purposes. METHODS: This study was designed to examine whether the preconditioning treatment with hypoxia or nitric oxide could trigger biogenesis in undifferentiated and differentiated neuronal cells (rat PC12 and human NT2 cells) as well as in primary mouse cortical neurons. RESULTS: The results showed that both preconditioning paradigms induced mitochondrial biogenesis in undifferentiated cell lines, as indicated by an increase of mitochondrial mass (measured by flow cytometry of NAO fluorescence) and increased expression of genes required for mitochondrial biogenesis (Nrf1, Nrf2, Tfam, Nfκb1) and function (Cox3, Hk1). All these changes translated into an increase in the organelle copy number from an average of 20-40 to 40-60 mitochondria per cell. The preconditioning treatments also rendered the cells significantly less sensitive to the subsequent oxidative stress challenge brought about by oxygen/glucose deprivation, consistent with their improved cellular energy status. Mitochondrial biogenesis was abolished when preconditioning treatments were performed in the presence of antioxidants (vitamin E or CoQ10), indicating clearly that ROS-signaling pathway(s) played a critical role in the induction of this phenomenon in undifferentiated cells. However, mitochondrial biogenesis could not be re-initiated by preconditioning treatments in any of the post-mitotic neuronal cells tested, i.e., neither rat PC12 cells differentiated with NGF, human NT2 cells differentiated with retinoic acid nor mouse primary cortical neurons. Instead, differentiated neurons had a much higher organelle copy number per cell than their undifferentiated counterparts (100-130 mitochondria per neuron vs. 20-40 in proliferating cells), and this feature was not altered by preconditioning. CONCLUSIONS: Our study demonstrates that mitochondrial biogenesis occurred during the differentiation process resulting in more beneficial energy status and improved tolerance to oxidative stress in neurons, putting in doubt whether additional enhancement of this phenomenon could be achieved and successfully exploited as a way for better neuroprotection.

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2.

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 delta isoform (PP1cdelta) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.

Ubisol-Q10, a Nanomicellar and Water-Dispersible Formulation of Coenzyme-Q10 as a Potential Treatment for Alzheimer's and Parkinson's Disease.

The world continues a desperate search for therapies that could bring hope and relief to millions suffering from progressive neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD). With oxidative stress thought to be a core stressor, interests have long been focused on applying redox therapies including coenzyme-Q10. Therapeutic use has failed to show efficacy in human clinical trials due to poor bioavailability of this lipophilic compound. A nanomicellar, water-dispersible formulation of coenzyme-Q10, Ubisol-Q10, has been developed by combining coenzyme-Q10 with an amphiphilic, self-emulsifying molecule of polyoxyethanyl α-tocopheryl sebacate (derivatized vitamin E). This discovery made possible, for the first time, a proper assessment of the true therapeutic value of coenzyme-Q10. Micromolar concentrations of Ubisol-Q10 show unprecedented neuroprotection against neurotoxin exposure in in vitro and in vivo models of neurodegeneration and was extremely effective when delivered either prior to, at the time of, and most significantly, post-neurotoxin exposure. These findings indicate a possible way forward for clinical development due to effective doses well within Federal Drug Administration guidelines. Ubisol-Q10 is a potent mobilizer of astroglia, antioxidant, senescence preventer, autophagy activator, anti-inflammatory, and mitochondrial stabilizer. Here we summarize the work with oil-soluble coenzyme-Q10, its limitations, and focus mainly on efficacy of water-soluble coenzyme-Q10 in neurodegeneration.

Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells.

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 microM ethanol or 20 microM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity.

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters. The overexpression of GDNF, regardless of the promoter employed, had no obvious adverse effects on astroglia and the engineered cells stably produced and secreted GDNF for extended periods of time (> or =3 weeks). These astrocytes very effectively protected neurons against 6OHDA, in both mouse and human co-culture systems. The neuroprotective effects were mediated not only by GDNF, but also by the antioxidant GSH since its depletion reduced the level of GDNF protection. Furthermore, neurons and astrocytes expressed different components of GDNF signaling complex, suggesting that they might utilize separate pathways to mediate autocrine and paracrine effects of GDNF.

Paraquat induces oxidative stress, neuronal loss in substantia nigra region and parkinsonism in adult rats: neuroprotection and amelioration of symptoms by water-soluble formulation of coenzyme Q10.

BACKGROUND: Parkinson's disease, for which currently there is no cure, develops as a result of progressive loss of dopamine neurons in the brain; thus, identification of any potential therapeutic intervention for disease management is of a great importance. RESULTS: Here we report that prophylactic application of water-soluble formulation of coenzyme Q10 could effectively offset the effects of environmental neurotoxin paraquat, believed to be a contributing factor in the development of familial PD. In this study we utilized a model of paraquat-induced dopaminergic neurodegeneration in adult rats that received three weekly intra-peritoneal injections of the herbicide paraquat. Histological and biochemical analyses of rat brains revealed increased levels of oxidative stress markers and a loss of approximately 65% of dopamine neurons in the substantia nigra region. The paraquat-exposed rats also displayed impaired balancing skills on a slowly rotating drum (rotorod) evidenced by their reduced spontaneity in gait performance. In contrast, paraquat exposed rats receiving a water-soluble formulation of coenzyme Q10 in their drinking water prior to and during the paraquat treatment neither developed neurodegeneration nor reduced rotorod performance and were indistinguishable from the control paraquat-untreated rats. CONCLUSION: Our data confirmed that paraquat-induced neurotoxicity represents a convenient rat model of parkinsonian neurodegeneration suitable for mechanistic and neuroprotective studies. This is the first preclinical evaluation of a water-soluble coenzyme Q10 formulation showing the evidence of prophylactic neuroprotection at clinically relevant doses.

Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation.

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell-type-specific epigenomes and suggest mechanisms by which they may be created and maintained.

Ubisol-Q10 (a Nanomicellar Water-Soluble Formulation of CoQ10) Treatment Inhibits Alzheimer-Type Behavioral and Pathological Symptoms in a Double Transgenic Mouse (TgAPEswe, PSEN1dE9) Model of Alzheimer's Disease.

 Alzheimer's disease (AD) is one of the most common neurodegenerative pathologies for which there are no effective therapies to halt disease progression. Given the increase in the incidence of this disorder, there is an urgent need for pharmacological intervention. Unfortunately, recent clinical trials produced disappointing results. Molecular mechanisms of AD are converging on the notion that mitochondrial dysfunction, oxidative stress, and accumulation of dysfunctional proteins are involved in AD pathology. Previously, we have shown that a water-soluble formulation of Coenzyme Q10 (Ubisol-Q10), an integral part of the electron transport chain, stabilizes mitochondria and prevents neuronal cell death caused by neurotoxins or oxidative stress both in vitro and in vivo. In this study, we evaluated the neuroprotective effects of Ubisol-Q10 treatment in double transgenic AD mice. In the present study, we report that providing Ubisol-Q10 in drinking water (at a dose of ∼6 mg/kg/day) reduced circulating amyloid-ß (Aß) peptide, improved long term memory, preserved working spatial memory, and drastically inhibited Aß plaque formation in 18-month-old transgenic mice compared to an untreated transgenic group. Thus Ubisol-Q10 supplementation has the potential to inhibit the progression of neurodegeneration, leading to a better quality of life for humans suffering with AD.

Evolution of motif variants and positional bias of the cyclic-AMP response element.

BACKGROUND: Transcription factors regulate gene expression by interacting with their specific DNA binding sites. Some transcription factors, particularly those involved in transcription initiation, always bind close to transcription start sites (TSS). Others have no such preference and are functional on sites even tens of thousands of base pairs (bp) away from the TSS. The Cyclic-AMP response element (CRE) binding protein (CREB) binds preferentially to a palindromic sequence (TGACGTCA), known as the canonical CRE, and also to other CRE variants. CREB can activate transcription at CREs thousands of bp away from the TSS, but in mammals CREs are found far more frequently within 1 to 150 bp upstream of the TSS than in any other region. This property is termed positional bias. The strength of CREB binding to DNA is dependent on the sequence of the CRE motif. The central CpG dinucleotide in the canonical CRE (TGACGTCA) is critical for strong binding of CREB dimers. Methylation of the cytosine in the CpG can inhibit binding of CREB. Deamination of the methylated cytosines causes a C to T transition, resulting in a functional, but lower affinity CRE variant, TGATGTCA. RESULTS: We performed genome-wide surveys of CREs in a number of species (from worm to human) and showed that only vertebrates exhibited a CRE positional bias. We performed pair-wise comparisons of human CREs with orthologous sequences in mouse, rat and dog genomes and found that canonical and TGATGTCA variant CREs are highly conserved in mammals. However, when orthologous sequences differ, canonical CREs in human are most frequently TGATGTCA in the other species and vice-versa. We have identified 207 human CREs showing such differences. CONCLUSION: Our data suggest that the positional bias of CREs likely evolved after the separation of urochordata and vertebrata. Although many canonical CREs are conserved among mammals, there are a number of orthologous genes that have canonical CREs in one species but the TGATGTCA variant in another. These differences are likely due to deamination of the methylated cytosines in the CpG and may contribute to differential transcriptional regulation among orthologous genes.

Novel subtractive transcription-based amplification of mRNA (STAR) method and its application in search of rare and differentially expressed genes in AD brains.

BACKGROUND: Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR), is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes. RESULTS: Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner). CONCLUSION: The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.

Effects of nitric oxide donors on cybrids harbouring the mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) A3243G mitochondrial DNA mutation.

Reactive nitrogen and oxygen species (O2*-, H2O2, NO* and ONOO-) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243-->Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential. However, endogenous levels of reactive oxygen species were very similar in all cybrids regardless of whether they carried the mtDNA defects or not. Furthermore, the cybrids were insensitive to the nitrosative and/or nitrative stress produced by either DETA-NO or SIN-1 alone. Cytotoxicity, however, was observed in response to SNP treatment and a combination of SIN-1 and glucose-deprivation. The mutant cybrids were significantly more sensitive to these insults compared with the wt controls. Ultrastructural examination of dying cells revealed several characteristic features of autophagic cell death. We concluded that nitrosative and/or nitrative stress alone were insufficient to trigger cytotoxicity in these cells, but cell death was observed with a combination of metabolic and nitrative stress. The vulnerability of the cybrids to these types of injury correlated with the cellular energy status, which were compromised by the MELAS mutation.

Identification of a functional CRE in the promoter of Fukuyama congenital muscular dystrophy gene fukutin.

We isolated a fragment of the fukutin gene promoter from differentiated human NT2 cells using chromatin immunoprecipitation technique with an anti-CREB antibody. This fragment contained a CRE-like sequence and here we describe its functional validation. The results showed that the element was functional in vitro and in vivo and that CREB in neurons was involved in the transcriptional regulation of the fukutin gene. Moreover, its expression in neurons was regulated by cAMP and calcium ions, known triggers of CREB phosphorylation. To our knowledge, this is the first report on the regulation of fukutin gene by transcription factor CREB in response to the signals generated by synaptic activity. The true biological function of fukutin, the gene responsible for Fukuyama-type congenital muscular dystrophy and mental retardation, is at present not known. However, it has been suggested that it might possess glycosyltransferase activity and its intracellular localization within the Golgi structures is consistent with this function. As such, fukutin might play a significant role in post-translational modification of synaptic proteins in neuronal cells.

Attenuation of UV-induced apoptosis by coenzyme Q10 in human cells harboring large-scale deletion of mitochondrial DNA.

Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial diseases caused by large-scale deletions in mitochondrial DNA (mtDNA) that impair the respiratory function of mitochondria and result in decreased production of ATP in affected tissues. In order to investigate whether CPEO-associated mtDNA mutations (i.e., 4,366-bp and 4,977-bp large-scale deletions) render human cells more vulnerable to apoptosis, we constructed cybrids carrying the deleted mtDNA. Assays for cell viability, DNA fragmentation, cytochrome c release, and caspase 3 activation revealed that UV irradiation at 20 J/m2 triggered apoptosis in all the cybrids. This treatment also produced elevated intracellular levels of reactive oxygen species (ROS). The rate of UV-induced cell death was more pronounced in the cybrids harboring mtDNA deletions than in the control cybrid with wild-type mtDNA. Subsequently, we evaluated the effect of coenzyme Q10 on the UV-triggered apoptosis. The results showed that after pretreatment of the cybrids with 100 microM coenzyme Q10 the UV-induced cell damage (i.e., ROS production and activation of caspase 3) was significantly reduced. Taken together, these findings suggest that large-scale deletions of mtDNA increased the susceptibility of human cells to the UV-triggered apoptosis and that coenzyme Q10 mitigated the damage; hence, it might potentially serve as a therapeutic agent to treat mitochondrial diseases resulting from mtDNA deletions.

Neuroprotective effects of coenzyme Q10 at rostral ventrolateral medulla against fatality during experimental endotoxemia in the rat.

Coenzyme Q10 (CoQ10, ubiquinone) is a highly mobile electron carrier in the mitochondrial respiratory chain that also acts as an antioxidant. We evaluated the neuroprotective efficacy of CoQ10 against fatality in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome using a novel water-soluble formulation of this quinone derivative. Experiments were conducted in adult male Sprague-Dawley rats that were maintained under propofol anesthesia. Intravenous administration of Escherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced progressive hypotension, with death ensuing within 4 h. The sequence of cardiovascular events during this LPS-induced endotoxemia can be divided into a reduction (Phase I), followed by an augmentation (Phase II; "pro-life" phase) and a secondary decrease (Phase III; "pro-death" phase) in the power density of the vasomotor components (0-0.8 Hz) of systemic arterial pressure signals. Pretreatment by microinjection bilaterally of CoQ10 (1 or 2 microg) into the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, significantly diminished mortality, prolonged survival time, and reduced the slope or magnitude of the LPS-induced hypotension. CoQ10 pretreatment also significantly prolonged the duration of and augmented the total power density of the vasomotor components of systemic arterial pressure signals in Phase II endotoxemia. The increase in superoxide anion production induced by LPS at the RVLM during Phases II and III endotoxemia was also significantly blunted. We conclude that CoQ10 provides neuroprotection against fatality during experimental endotoxemia by reducing superoxide anion production at the RVLM, whose neuronal activity is intimately related to the "life-and-death" process.

Identification of functional dopamine receptors in human teratocarcinoma NT2 cells.

In search of a cellular model suitable for studying molecular events contributing to brain disorders, we have characterised the expression and functionality of dopamine receptors in human teratocarcinoma NT2 cells. The cells were differentiated by a 4-week retinoic acid treatment, followed by a 3-week mitotic inhibitor treatment in the absence of retinoic acid. The messages of two D(2)-like family members, D(2L) and D(3), were expressed in undifferentiated NT2 cells. The retinoic acid treatment resulted in increased expression of both spliced variants of the D(2) receptor, D(2L) and D(2S) isoforms and a significant induction of D(1) and D(5) gene transcripts. The same treatment turned off expression of the D(3) gene. Further induction of the D(5) gene was observed in the post-mitotic NT2N neurons. The NT2N cells stained positively for D(2) and D(5) receptor proteins, and the intracellular cyclic AMP level increased in response to forskolin, dopamine and the D(1)-receptor agonist SKF-81297. Furthermore, dopamine was ineffective in the presence of the D(2) receptor agonist PPHT and the D(1) receptor antagonist cis-(z)-flupenthixol. These results indicated that upon ligand/agonist/antagonist binding, the receptors could be coupled to the adenylyl cyclase system, hence were functional. To our knowledge, NT2 is the only human immortalized cell line expressing functional dopamine receptors of both families.

Measurement of apoptosis by DNA fragmentation.

Classical apoptotic cell death can be defined by certain morphological and biochemical characteristics that distinguish it from other forms of cell death. One such feature, which is a hallmark of apoptosis, is DNA fragmentation. In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180-bp oligomers and appear as a DNA ladder when run on an agarose gel. Here, we present commonly used methods such as conventional agarose gel electrophoresis to analyze fragmented nuclei in cells. The various methods used are dependent on the extent of fragmentation or the amount of fragmented nuclei in a sample. Determining whether a cell exhibits DNA fragmentation can provide information about the type of cell death occurring and the pathways activated in the dying cell.

Unique technology for solubilization and delivery of highly lipophilic bioactive molecules.

We have produced a family of novel carriers enabling water solubilization of highly lipophilic molecules. The compound carriers were synthesized by conjugating polyethylene glycol to alpha-tocopherol, tocotrienols, beta-sitosterol or cholesterol via an alkanedioyl linker. These PEG- conjugates were amphiphilic and formed stable non-covalent complexes (nanomicelles) with a wide range of molecules including vitamins, carotenoids, ubiquinones, poly-unsaturated fatty acids and polyene macrolide antibiotics. The resulting formulations were water-soluble, non-toxic and had excellent stability. This solubilization method represents a major advance in the delivery of lipophilic molecules and could be used to reformulate drugs with near term patent expiry or those that have failed clinical trials due to low solubility. Furthermore, the technology could also be applied for delivery of active ingredients for dietary supplement, functional food, cosmetic and animal health industries.